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Mutations M287L and Q266I in the ␣1 Subunit Change Sensitivity to Volatile Anesthetics in Oocytes and , but Not the Minimal Alveolar Concentration in Knockin Mice

Cecilia M. Borghese, Ph.D.,* Wei Xiong, Ph.D.,† S. Irene Oh, B.S.,‡ Angel Ho, B.S.,§ S. John Mihic, Ph.D.,ʈ Li Zhang, M.D.,# David M. Lovinger, Ph.D.,** Gregg E. Homanics, Ph.D.,†† Edmond I. Eger 2nd, M.D.,‡‡ R. Adron Harris, Ph.D.§§ Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/117/4/765/257527/0000542-201210000-00019.pdf by guest on 01 October 2021

ABSTRACT What We Already Know about This Topic • Inhibitory spinal function is enhanced by vol- Background: Volatile anesthetics (VAs) alter the function of atile anesthetics, making this a leading candidate for their key proteins but it is not clear which, immobilizing effect • ␣ if any, of these targets mediates the immobility produced by Point mutations in the 1 subunit of glycine receptors have been identified that increase or decrease receptor potentiation VAs in the face of noxious stimulation. A leading candidate is by volatile anesthetics the glycine receptor, a -gated important for spinal physiology. VAs variously enhance such function, and blockade of spinal glycine receptors with af- fects the minimal alveolar concentration (an anesthetic What This Article Tells Us That Is New • EC50) in proportion to the degree of enhancement. Mice harboring specific mutations in their glycine receptors Methods: We produced single mutations into that increased or decreased potentiation by volatile anesthetic ␣ in vitro did not have significantly altered changes in anesthetic the glycine receptor 1 subunit that increased (M287L, third potency in vivo transmembrane region) or decreased (Q266I, second trans- • These findings indicate that this glycine receptor does not me- membrane region) sensitivity to in recombinant diate anesthetic immobility, and that other targets must be receptors, and introduced such receptors into mice. The re- considered sulting knockin mice presented impaired trans- mission, but heterozygous animals survived to adulthood, * Research Associate, ʈ Associate Professor, §§ Professor, The and we determined the effect of isoflurane on glycine-evoked University of Texas at Austin, Waggoner Center for and responses of brainstem neurons from the knockin mice, and Addiction Research, Austin, Texas. † Research Fellow, # Medical Officer, ** Laboratory Chief, Laboratory for Integrative Neurosci- the minimal alveolar concentration for isoflurane and other ence, National Institute on Alcohol Abuse and Alcoholism, National VAs in the immature and mature knockin mice. Institutes of Health, Rockville, Maryland. ‡ Staff Research Associate, Results: Studies of glycine-evoked currents in brainstem ‡‡ Professor Emeritus, Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, Califor- neurons from knockin mice confirmed the changes seen with nia. § Staff Research Assistant, Diabetes Center, University of Cali- recombinant receptors. No increases in the minimal alveolar fornia San Francisco. †† Professor, Departments of Anesthesiology concentration were found in knockin mice, but the minimal and Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania. alveolar concentration for isoflurane and (but not Received from the Waggoner Center for Alcohol and Addiction ) decreased in 2-week-old Q266I mice. This Research, The University of Texas at Austin, Austin, Texas. Submit- change is opposite to the one expected for a mutation that ted for publication October 10, 2011. Accepted for publication June decreases the sensitivity to volatile anesthetics. 7, 2012. Supported by grant nos. 1PO1GM47818 and AA06399 from the National Institutes of Health, Bethesda, Maryland. Drs. Xiong, Conclusion: Taken together, these results indicate that gly- Zhang, and Lovinger acknowledge support from the Division of cine receptors containing the ␣1 subunit are not likely to be Intramural Clinical and Biomedical Research, National Institute on crucial for the action of isoflurane and other VAs. Alcohol Abuse and Alcoholism, National Institutes of Health, Rock- ville, Maryland. Address correspondence to Dr. Harris: The University of PRESENT consensus suggests that volatile anesthetics Texas at Austin, Waggoner Center for Alcohol and Addiction (VAs) bind to proteins to produce their effects, includ- Research, 1 University Station A4800, Austin, Texas 78712-0159. A 1 [email protected]. Information on purchasing reprints may ing immobility. The key question is, “Which proteins?” The be found at www.anesthesiology.org or on the masthead page at list of candidates is short.1–3 A top candidate is the glycine the beginning of this issue. ANESTHESIOLOGY’s articles are made receptor (GlyR). freely accessible to all readers, for personal use only, 6 months from the cover date of the issue. Evidence for the GlyR as a target for VA includes their Copyright © 2012, the American Society of Anesthesiologists, Inc. Lippincott localization and sensitivity to VAs. The RNAs encoding the 4 Williams & Wilkins. Anesthesiology 2012; 117:765–71 GlyR subunits are mostly in the and brainstem,

Anesthesiology, V 117 • No 4 765 October 2012 Glycine Receptors and Anesthesia

and the spinal cord mediates the immobility effect of VA.5 in 50% of test subjects) of anesthetics in wild-type and Moreover, anesthetic concentrations of VAs substantially en- knockin mice. hance inhibitory currents through the GlyR, both in heter- ologous systems6–8 and neurons.8 More direct evidence Materials and Methods arises from the use of strychnine, a competitive GlyR-specific Knockin mouse production and the electrophysiological antagonist, both at the neuronal and whole animal level. The techniques were described in detail in Borghese et al.21 We application of halothane to decerebrate rats depressed the briefly describe them here, and add pertinent details. The spinal cord sensory neuronal activity to noxious stimuli; experimental work on animals conformed to the guidelines the concurrent application of strychnine partially reversed laid out in the Guide for the Care and Use of Laboratory the halothane-induced depression.9 reduced Animals, and approval of the corresponding institutional spontaneous firing of ventral horn interneu- Animal Care Committees was obtained. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/117/4/765/257527/0000542-201210000-00019.pdf by guest on 01 October 2021 rons in cultured spinal cord tissue slices; application of Electrophysiology in Xenopus Oocytes strychnine decreased the sevoflurane effect.10 Intrathecal ad- was purchased from Sigma-Aldrich (Saint ministration of strychnine increased the minimal alveolar Louis, MO), and isoflurane was purchased from Marsam concentration (MAC) of isoflurane.11 The magnitude of Pharmaceuticals (Cherry Hill, NJ). Isoflurane and chloro- GlyR potentiation expressed in oocytes varies according to 12 form solutions were prepared in buffer immediately before the VA used. Intrathecal administration of strychnine to application. rats anesthetized with halothane, isoflurane, or Manually isolated oocytes from Xenopus laevis frogs were increased MAC in proportion to the enhancement of GlyRs injected in the nuclei with complementary DNA encoding observed in vitro13. Furthermore, intrathecal glycine reduced ␣ 14 wild-type and mutated human 1 GlyR subunits. After 1–5 the isoflurane MAC in rats. These results are compatible days, recordings were carried out using the whole cell two- with the hypothesis that GlyRs partly mediate the capacity of electrode voltage clamp configuration. All drugs were ap- VAs to produce immobility. plied by bath-perfusion and solutions were prepared the day ␥ Construction of chimeric glycine- -aminobutyric acid of the experiment. To study the chloroform (1 mM) and type A receptors (GABAA-Rs) allowed the identification of isoflurane (0.3 mM) modulation of glycine currents, the gly- transmembrane amino acids crucial to the VAs’ action, cine concentration equivalent to 5% of the maximal glycine- namely S267 and A288 in the second and third GlyR trans- evoked current (EC5) was determined after applying the gly- 15 membrane domains. Mutation of S267 to cysteine and cine concentration that produced maximal current (3 mM for subsequent labeling with thiol-specific reagents blocked the wild-type, 10 mM for mutant). Glycine was removed with a VA potentiation of GlyRs.16,17 Furthermore, a crystal struc- washout of 5 min between all applications, except after ap- ture of a prokaryotic protein closely related to GlyR was plication of maximal glycine concentrations (15 min). After obtained in the presence of VAs and showed selective bind- two applications of EC5 glycine, each of the modulators was ing of the anesthetics to the regions proposed in the earlier preapplied for 1 min and then coapplied with glycine for 18 studies of GlyR. 30 s. EC5 glycine was applied between coapplication of gly- The identification of a mutation in a receptor that renders cine and modulator. The concentrations of isoflurane and it insensitive or hypersensitive to modulation by VAs would chloroform were chosen as equivalent to1xMAC.22 All allow construction of a knockin mouse that could be tested experiments shown include data obtained from oocytes for changes in the sensitivity to VAs. This approach was taken from at least two different frogs, and oocytes that pre- sented a maximal current more than 20 ␮A were not in- successful in showing that specific GABAA-Rs are responsi- ble for at least some of the actions of certain injectable anes- cluded in the data collection. thetics.19,20 In our studies of alcohol action, we found two Knockin Mouse Production mutations in the ␣1 GlyR subunit, one in the second Breeding colonies were established in the Lovinger and Eger (Q266I), and another in the third transmembrane domain laboratories, and mice were produced by heterozygous breed- (M287L) that decreased modulation of GlyR.21 Mu- ing, resulting in litters with wild-type, heterozygous mutant, tant mice were constructed in which each of these mutant ␣1 and homozygous mutant mice. Homozygous mice for either GlyR subunits replaced the endogenous GlyR subunit 21 mutation appear normal at birth, but display and (knockin mice). The goal of the present study was to de- reduced growth by 2 weeks of age with lethality occurring by termine if these mutations altered the action of VA on GlyR 2–3 weeks of age for Q266I mice.21 Thus, studies with mice and if such alterations would affect the immobility produced older than 3 weeks of age were carried out with only by VA. This was accomplished by studying glycine responses heterozygous (. wild-type) mice. of recombinant receptors expressed in Xenopus oocytes and of neurons isolated from brainstem of wild-type and knockin Isolation of Neurons and Electrophysiological Recording mice, and the immobilizing potency (the MAC of anesthetic Postnatal day 14–21 mice were sacrificed by decapitation that abolished movement in response to noxious stimulation during halothane (Sigma-Aldrich) anesthesia and their

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brains were rapidly removed and placed in ice-cold buffer. mice. Mice then were returned to their home cages and in 2 Brainstem slices were cut using a vibrating blade microtome days tested with another anesthetic. The test anesthetics were (Leica VT1200S; Leica Microsystems, Wetzlar, Germany), isoflurane and enflurane for M287L mice, and isoflurane, incubated with pronase, and then with thermolysin. The halothane, and enflurane for Q266I mice. Surviving mice slices were then transferred to a culture dish, and mildly (i.e., wild-type and heterozygous mice) then were allowed to triturated using pipettes to dissociate single neurons. grow to 8–12 weeks of age and were tested for MAC for Membrane currents were recorded in the whole cell con- isoflurane, enflurane, and/or cyclopropane. figuration at 20–22°C. Cells were held at Ϫ60 mV unless The chromatograph (Gow-Mac 750 flame ionization otherwise indicated. Data were acquired using pClamp 9.2 detector gas chromatograph; Gow-Mac Instrument Corp., software (Molecular Devices, Sunnyvale, CA). Isoflurane Bridgewater, NJ) was calibrated with secondary standards was purchased from Baxter (Deerfield, IL). Isoflurane solu- from tanks containing volatile anesthetic or from concentra- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/117/4/765/257527/0000542-201210000-00019.pdf by guest on 01 October 2021 tion was prepared in the extracellular recording solution im- tions obtained by serial dilution from 100% cyclopropane. mediately before beginning experiments each day, and kept MAC for each mouse was calculated as the mean of the in a sealed bottle. Solutions were applied through 3-barrel concentrations that bracketed movement-nonmovement. square glass tubing (Warner Instrument, Hamden, CT) with The group MAC and standard errors were calculated from a tip diameter of approximately 700 ␮m, using the Warner these individual values. Fast-Step stepper-motor driven system. The solution ex- change time constants were approximately 4 ms for an open Statistical Analysis pipette tip and 4–12 ms for whole cell recording. The solu- Prism (GraphPad Software Inc., La Jolla, CA) was used to tion in the wells feeding into the solution exchange system conduct the statistical analysis. In all cases, pooled data are was changed after recording from 1 or 2 cells to replenish presented as mean Ϯ SEM. Differences were assessed with isoflurane in the line. The drug application system was sealed two-tailed Student t test in the oocyte recordings. In the at all times except when applying isoflurane to a cell, to neuronal recordings, we used one-way ANOVA followed by reduce loss of the volatile anesthetic. In neurons that were Dunnett multiple comparison test, where the objective is to recorded for at least 30 min, the isoflurane-induced potenti- identify groups whose means are significantly different from ation did not change over time, indicating limited, if any, loss the mean of a selected “reference group,” in our case, the of the volatile anesthetic from our system. wild-type mice. We accepted P Ͻ 0.05 as significant.

MAC Determination Results Animals were held in rooms with 12-h on-off light cycles. We first asked if the M287L or Q266I mutations altered the The offspring were observed daily for signs of central nervous sensitivity of GlyR to modulation by anesthetics. The appli- system irritability, namely tremors. When tremors were ob- cation of isoflurane or chloroform produced different effects served in a given litter, the capacity of VA to induce immo- on the glycine-evoked currents through the mutant receptors bility in 50% of individual littermates in the face of noxious expressed in Xenopus oocytes (representative tracings in the stimulation (i.e., MAC) was tested within 2–4 days for all presence of isoflurane in fig. 1). When applied to the 23 mice using techniques described previously. Rectal tem- ␣1(M287L) GlyR, 0.3 mM isoflurane showed an increased peratures were monitored and usually maintained between potentiation compared with ␣1 wild-type, whereas the chlo- 35.5 and 38.5°C. Anesthetic concentrations were monitored roform (1 mM) potentiation was unchanged (fig. 2A). In line with an infrared analyzer (Datascope, Helsinki, Finland). with the observed lack of alcohol effects on Q266I,21 isoflu- The initial anesthetic concentration was imposed for a 40- rane potentiation was almost abolished in ␣1(Q266I) GlyR, min equilibration period. A low initial target concentration and chloroform potentiation was severely decreased (fig. 2B). was chosen to ensure movement in response to up to a 1-min Previous studies had shown that although the glycine af- application of an alligator clip to the base of the tail. We then finity is not modified in neurons from homozygous mice, the increased the anesthetic concentration in steps of 15–20% of maximal glycine current is decreased in homozygous mice.21 the preceding concentration, holding each step for a mini- We studied the isoflurane effect on submaximal (10 ␮M) mum of 20 to 30 min before again applying the alligator clip. glycine-activated currents in isolated neurons from the This continued until all mice ceased moving in response to knockin mice (fig. 3 shows representative tracings). The the stimulation. At the end of each step (after application GlyR modulation by isoflurane (1 mM) in isolated neurons of the tail clip), a sample of gas was taken for anesthetic from knockin mice showed changes that were similar to analysis by gas chromatography. On completion of these those observed in Xenopus oocytes. In neurons from M287L studies, snips of tail tips were sent to the Harris laboratory for mice, the isoflurane effect on glycine-evoked currents was genotyping. Two groups that were not genotyped (2-week- increased, and this depended on gene “dosage”: it was greater old Q266I mice tested with enflurane and halothane) were in neurons from homozygous than in heterozygous mice, grouped according to the phenotype: tremoring (homozy- and greater in heterozygous than in wild-type mice (figs. 3A gous) and non-tremoring (wild-type and heterozygous) and fig. 4). In neurons from Q266I mice, the isoflurane

Anesthesiology 2012; 117:765–71 767 Borghese et al. Glycine Receptors and Anesthesia Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/117/4/765/257527/0000542-201210000-00019.pdf by guest on 01 October 2021

Fig. 1. Representative tracings of isoflurane (0.3 mM) effect on currents induced by glycine (with a concentration that induced ␣ ␣ ␣ a current that is 5% of the maximal current, effective concentration 5, or EC5) in homomeric 1 wild-type, 1 (M287L) and 1 (Q266I) GlyRs expressed in Xenopus oocytes. Data from multiple recordings are presented in fig. 2. WT ϭ wild-type. effect was decreased according to the presence of the muta- for isoflurane and cyclopropane were not modified com- tion, and essentially absent in homozygous and smaller in pared with wild-type mice. In 2-week-old Q266I mice, heterozygous than in wild-type (figs. 3B and 4B). tremoring (homozygous) mice showed a decrease in isoflu- Anesthetic sensitivity in vivo was evaluated by determin- rane and enflurane MAC values compared with non-tremo- ing the MAC values for M287L and Q266I mice of different ring (heterozygous and wild-type), whereas halothane ages: 14–21 days of age and adult (more than 8 weeks old), in showed no differences between these two groups. homozygous, heterozygous, and wild-type mice (tables 1 and 2). Cyclopropane was tested as a negative control; it has a Discussion small effect on GlyRs expressed in heterologous systems.12 Defining protein sites of VA action has proven remarkably The MAC values for isoflurane and enflurane were un- difficult.1,3 It is useful to consider the criteria that must be changed in M287L knockin mice compared with wild-type satisfied for a protein to be considered a plausible target for mice. In adult Q266I heterozygous mice, the MAC values VA action. VAs have many similarities to ethanol (small mol- ecules, low potency, multiple potential sites of action), and ethanol might be considered as a VA. Four criteria were proposed for defining a molecular target of alcohol action,24 and they are equally applicable to other VA. In brief, they are: (1) function of the protein must be altered by pharmacolog-

Fig. 2. Volatile anesthetics’ effect on glycine responses in homomeric ␣1 wild-type (WT), ␣1 (M287L), and ␣1 (Q266I) Fig. 3. Representative tracings of isoflurane (1 mM)on(10␮M) expressed in Xenopus oocytes. Isoflurane (0.3 mM) and chlo- glycine-evoked currents in neurons from wild-type or roform (1 mM) potentiation of glycine responses (with a con- heterozygous mutant mice. (A) Currents in neurons from ␣1 centration that induced a current that is 5% of the maximal (M287L) mice: ␣1M/M, wild-type (upper panel) and ␣1M/L, ␣ ␣ current, effective concentration 5, or EC5)in(A) 1 wild-type heterozygous (lower panel). (B) Currents in neurons from 1 (n ϭ 6) and ␣1 (M287L) (n ϭ 5), and (B) ␣1 wild-type (n ϭ 4) (Q266I) mice: ␣1Q/Q, wild-type (upper panel) and ␣1Q/I, and ␣1 (Q266I) (n ϭ 4). Values are means Ϯ SEM; *P Ͻ 0.01 heterozygous (lower panel). Data from multiple tracings are versus corresponding wild-type, Student t test analysis. presented in Fig. 4. Gly ϭ glycine.

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where a knockin mouse with a GABAA-R resistant subunit [␤3(N265M)] displayed remarkable resistance to anesthet- ics20 with little change in the action of VAs.28 With the lack

of support for GABAA-R as a mechanism for the immobility produced by VA, GlyR emerged as one of the “last best hopes,”29 making the construction and study of knockin mice for this receptor a critical step. To accomplish this, we constructed single amino acid mutations in the ␣1 GlyR subunit that would increase or decrease the actions of VA and constructed knockin mice with each of the mutations.21 We chose the ␣1 subunit because there are two kinds of GlyR Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/117/4/765/257527/0000542-201210000-00019.pdf by guest on 01 October 2021 subunits, ␣ (␣1–4) and ␤. The ␣2 subunit predominates perinatally, whereas the heteromeric ␣1␤ GlyRs replace them during the first 3 weeks of the postnatal stage, and are the main GlyR present in spinal cord and brainstem in adult- hood.4 The ␣1␤ GlyR is difficult to express in Xenopus Fig. 4. Isoflurane (1 mM) effect on glycine-evoked (10 ␮M) oocytes, so we decided to use the homomeric ␣1 GlyR in our currents in isolated neurons from wild-type and knockin heterologous experiments. mice. (A) Neurons from M287L knockin mice: ␣1M/M, wild- ␣ M/L ␣ L/L The striking finding from the present experiments is that type; 1 , heterozygous; 1 , homozygous. (B) Neurons ␣ from Q266I knockin mice: ␣1Q/Q, wild-type; ␣1Q/I, heterozy- introduction of GlyR 1 subunits with either enhanced or gous; ␣1I/I, homozygous. Values are means Ϯ SEM; n ϭ 6 for decreased modulation by VA into mice has almost no effect each group; *P Ͻ 0.05, ***P Ͻ 0.001 versus corresponding on the ability of these agents to produce immobility, and wild-type, one-way ANOVA, Dunnett post hoc test. none in the direction expected if the immobility effect was mediated by enhancement of currents mediated by GlyRs. ically reasonable concentrations of the drug; (2) mutation of Electrophysiological studies of recombinant receptors as well specific amino acids should provide evidence of a binding as neurons from the mutant mice showed that the M287L site; (3) manipulation of the protein in vivo (e.g., knockin and Q266I mutations in transmembrane regions of the GlyR mice) should produce expected changes in behavioral actions consistently altered anesthetic actions, yet the MAC for im- of the drug; and (4) physical structural studies should dem- mobility was not altered for any anesthetic tested, with two onstrate the presence of the drug at the proposed sites in the exceptions. The 2-week-old homozygous Q266I mice protein. Both GABA -Rs and GlyRs fulfill several of these A showed a small decrease in the MAC value for isoflurane and criteria. Pharmacological concentrations of most (but not all) VAs enhance the function of these receptors,2 and extensive enflurane (17% and 9%, respectively). If the immobility ef- mutation and thiol-labeling studies provide evidence for a fect were in part mediated by GlyRs, the presence of a mu- binding site within the transmembrane regions.15–17 In ad- tation in the GlyRs that made them insensitive to a VA dition, a recent structural study of GLIC, a ligand-gated ion would be predicted to increase, not decrease, the concentra- tion of anesthetic necessary to achieve immobility in the face channel closely related to GlyR and GABAA-R, shows the binding of anesthetics in the site proposed based on muta- of a noxious stimulus. In this particular case, the MAC was tions.18 The requirement to introduce a VA-resistant protein reduced. In 2-week-old mice, the perinatal ␣2 GlyRs are 44 into a mouse and reduce the VA immobilizing effect in vivo being replaced by the ␣1␤ GlyRs, which carry the Q266I has proven to be the most difficult criterion to satisfy. Thus mutation in ␣1. These mutated GlyRs have a reduced func- 21 far, knockin mice constructed with VA resistant GABAA-Rs tionality that translates in modification in the behavior and have not shown any increase in MAC.25–28 This is in contrast response to nonglycinergic drugs.30 Some of these changes to injectable anesthetics such as and , could be attributed to the development of compensatory

Table 1. Minimal Alveolar Concentration in M287L Knockin Mice

␣1M/M ␣1M/L ␣1L/L Volatile Anesthetic Age (Wild-type) (Heterozygous) (Homozygous) Isoflurane 2–4 weeks 1.65 Ϯ 0.07 (5) 1.77 Ϯ 0.02 (4) 1.65 Ϯ 0.06 (7) Adult* 1.54 Ϯ 0.07 (6) 1.50 Ϯ 0.06 (10) — Enflurane 2–4 weeks 2.45 Ϯ 0.07 (5) 2.42 Ϯ 0.08 (8) 2.33 Ϯ 0.03 (9) Adult 2.28 Ϯ 0.07 (6) 2.38 Ϯ 0.05 (7) —

Values are in % atmosphere (average Ϯ SEM, n between parenthesis). * Mice were 8 weeks old or older.

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Table 2. Minimal Alveolar Concentration in Q266I Knockin Mice

␣1Q/Q ␣1Q/I ␣1I/I Volatile Anesthetic Age (Wild-type) (Heterozygous) (Homozygous) Isoflurane 2 weeks 1.79 Ϯ 0.07 (10) 1.48 Ϯ 0.03 (8)* Enflurane 2 weeks 3.55 Ϯ 0.09 (8) 3.23 Ϯ 0.11 (5)* Halothane 2 weeks 1.93 Ϯ 0.05 (9) 1.86 Ϯ 0.09 (9) Isoflurane Adult 1.69 Ϯ 0.06 (7) 1.73 Ϯ 0.07 (5) — Cyclopropane Adult 18.7 Ϯ 1.1 (7) 16.4 Ϯ 1.0 (5) —

Values are in % atmosphere (average Ϯ SEM, n between parenthesis). Two-week-old mice were grouped according to their phenotype

(tremoring mice: homozygous; non-tremoring mice: wild-type or heterozygous). Adult mice (8 weeks old or older) were grouped Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/117/4/765/257527/0000542-201210000-00019.pdf by guest on 01 October 2021 according to genotype. * P Ͻ 0.05 vs. corresponding control group, Student t test. changes. Furthermore, the adult heterozygous Q266I did pensatory changes in the structures mediating immobility. not differ from wild-type in their response to isoflurane. We conclude that actions of VAs on the ␣1 GlyR have little Because strychnine increased MAC for VAs in proportion or no role in the production of immobility by VAs. As to their ability to modulate GlyR (halothane more than iso- Thomas Henry Huxley said,32 we face once again “the great flurane more than cyclopropane), it appeared that actions of tragedy of Science – the slaying of a beautiful hypothesis by VAs on GlyR likely mediated at least part of immobility, an ugly fact.” We conclude that the known molecular targets making the GlyR a leading candidate target for VAs.13,29 of VAs are not able to account for the immobilizing actions However, the current findings provide strong evidence that of VAs, and a search for new targets is required. the ␣1 GlyR is not important for VA immobility. One caveat is that the effects of the mutations we studied were not lim- The authors thank Rebecca J. Howard, Ph.D., Postdoctoral Fellow, and Kathryn Ondricek, B.S., Undergrad Research Assistant, for con- ited to alterations in VA actions on the receptors, because structing the Q266I mutant complementary DNA, and Chelsea Geil, they also impaired the function of GlyRs. It is possible that B.S., Research Assistant, Brianne Patton, B.S., Research Associate, reduced glycinergic neurotransmission in the mutants could and Virginia Bleck, B.S., Research Associate, for their excellent technical assistance. All five worked at The University of Texas at lead to compensatory changes in other . Austin, Waggoner Center for Alcohol and Addiction Research, Aus- Indeed, we found that the sedative effects of several drugs tin, Texas. that do not affect GlyRs, such as and , were increased in both mutants.30 In addition, the changes in References the loss of righting reflex induced by flurazepam and ket- 1. Franks NP: Molecular targets underlying general anaesthesia. were more pronounced in Q266I than in M287L, as Br J Pharmacol 2006; 147(Suppl 1):S72–81 could be expected considering that the impairment in glycin- 2. Yamakura T, Bertaccini E, Trudell JR, Harris RA: Anesthetics and ion channels: Molecular models and sites of action. Annu ergic transmission, as assessed by the acoustic startle reflex Rev Pharmacol Toxicol 2001; 41:23–51 and the mortality of homozygous knockin mice, was more 3. Eger EI 2nd, Raines DE, Shafer SL, Hemmings HC Jr, Sonner marked in Q266I than in M287L.21,30 However, we need to JM: Is a new paradigm needed to explain how inhaled anes- keep in mind that immobility (assessed through MAC) and thetics produce immobility? Anesth Analg 2008; 107:832–48 4. Lynch JW: Native glycine receptor subtypes and their phys- sedation (assessed through loss of righting reflex) are medi- iological roles. Neuropharmacology 2009; 56:303–9 31 ated by different areas in the central nervous system. Fur- 5. Antognini JF, Carstens E: In vivo characterization of clinical thermore, the data do not support the simple explanation anaesthesia and its components. Br J Anaesth 2002; 89: that main systems, like the GABAergic and glutamatergic 156–66 6. Mascia MP, Machu TK, Harris RA: Enhancement of homo- transmission, have undergone changes to compensate for meric glycine receptor function by long-chain and a decrease in the glycinergic transmission, because the anaesthetics. Br J Pharmacol 1996; 119:1331–6 N-methyl-D-aspartic acid-induced tonic convulsions were 7. Harrison NL, Kugler JL, Jones MV, Greenblatt EP, Pritchett the same in heterozygous knockin mice versus wild-type,30 DB: Positive modulation of human ␥-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflu- and the binding was unchanged in Q266I rane. Mol Pharmacol 1993; 44:628–32 21 heterozygous mice in brainstem and spinal cord. So, even if 8. Downie DL, Hall AC, Lieb WR, Franks NP: Effects of inhala- other signaling systems changed, we would have to propose tional general anaesthetics on native glycine receptors in rat that these changes exactly offset the altered sensitivity of the medullary neurons and recombinant glycine receptors in Xenopus oocytes. Br J Pharmacol 1996; 118:493–502 GlyR to VAs in exactly the same structures, when the com- 9. Yamauchi M, Sekiyama H, Shimada SG, Collins JG: Halothane pensatory changes would presumably offset the decreased suppression of spinal sensory neuronal responses to noxious glycinergic transmission, not the changes in the sensitivity to peripheral stimuli is mediated, in part, by both GABA(A) and isoflurane. This seems unlikely because both the M287L and glycine receptor systems. ANESTHESIOLOGY 2002; 97:412–7 10. 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